U.S. patent number 6,200,791 [Application Number 09/125,374] was granted by the patent office on 2001-03-13 for method of purifying thrombin-like protease enzymes obtained from snake venom.
This patent grant is currently assigned to Knoll Aktiengesellschaft. Invention is credited to Margarete Schwarz, Wolfgang Zahn.
United States Patent |
6,200,791 |
Schwarz , et al. |
March 13, 2001 |
Method of purifying thrombin-like protease enzymes obtained from
snake venom
Abstract
A process for purifying thrombin-like proteases from snake
venoms is described, which consists in freeing the proteases from
impurities in three chromatographic steps: a) affinity or anion
exchange, b) adsorption onto a glass matrix at alkaline pH values,
and c) size exclusion gel or glass matrix at acidic pH values.
Inventors: |
Schwarz; Margarete (Deidesheim,
DE), Zahn; Wolfgang (Altrip, DE) |
Assignee: |
Knoll Aktiengesellschaft
(Ludwigshafen, DE)
|
Family
ID: |
7786490 |
Appl.
No.: |
09/125,374 |
Filed: |
August 17, 1998 |
PCT
Filed: |
February 18, 1997 |
PCT No.: |
PCT/EP97/00755 |
371
Date: |
August 17, 1998 |
102(e)
Date: |
August 17, 1998 |
PCT
Pub. No.: |
WO97/32015 |
PCT
Pub. Date: |
September 04, 1997 |
Foreign Application Priority Data
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Feb 26, 1996 [DE] |
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196 07 210 |
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Current U.S.
Class: |
435/212; 435/814;
435/815 |
Current CPC
Class: |
C12N
9/6418 (20130101); A61P 7/02 (20180101); Y10S
435/815 (20130101); Y10S 435/814 (20130101) |
Current International
Class: |
C12N
9/64 (20060101); C12N 009/48 () |
Field of
Search: |
;435/212,814,815 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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24 28 955 |
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Mar 1975 |
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DE |
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27 34 427 |
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Feb 1978 |
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DE |
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1094301 |
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Dec 1967 |
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GB |
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1177506 |
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Jan 1970 |
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GB |
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1293793 |
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Oct 1972 |
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GB |
|
Other References
Merck Index, 11 Ed. 1989, p. 664. .
Bonilla, "Defibrinating Enzyme from Timber Rattlesnake (Crotalus H.
Horridus) Venom: A Potential Therapeutic for Defibrination I.
Purification and Properties", Thromb. Res., 6(2), pp. 151-169, Feb.
1975..
|
Primary Examiner: Weber; Jon P.
Attorney, Agent or Firm: Keil & Weinkauf
Parent Case Text
This is a 371 of International application No. PCT/EP97/00755,
filed Feb. 18, 1997.
Claims
We claim:
1. A method of purifying a thrombin-like protease from snake venom
selected from the group consisting of ancrod and strongly basic
proteases, which method comprises
a) obtaining a solution comprising the protease;
b) loading the protease solution onto an affinity chromatography
matrix or an anion exchange resin;
c) eluting the protease in an eluate solution from the affinity
chromatography matrix or anion exchange resin;
d) loading the eluate solution from step c) onto a matrix of glass
beads; said glass beads having a pore diameter of from 25 to 35 nm
and a particle size of from 30 to 60 .mu.m; said eluate being
applied to the matrix of glass beads in a basic solution at a pH
value of from 7.5 to 9.0 whereby the glass beads adsorb the
thrombin-like protease from snake venom;
e) eluting the protease in an eluate solution from the matrix of
glass beads;
f) loading the eluate solution from step e) onto a size exclusion
gel matrix or onto a matrix of glass beads; said glass beads having
a pore diameter of from 25 to 35 nm and a particle size of from 30
to 60 .mu.m; said eluate being applied to the matrix of glass beads
in an acidic solution; and
g) eluting and recovering the purified protease.
2. The method of claim 1, wherein the purification in step f) is
carried out using a size exclusion gel matrix.
3. The method of claim 1, wherein the eluate solution is loaded
onto a matrix of glass beads in step f).
4. The method of claim 1 in which the snake venom is from a snake
of the genus Agkistrodon.
5. The method of claim 4 in which the snake venom is from
Agkistrodon rhodostoma.
6. The method of claim 1 wherein the purified product is ancrod.
Description
The present invention relates to a process for purifying
thrombin-like proteases from snake venoms.
Examples of such proteases are batroxobin, crotalase and, in
particular, ancrod. The latter is an anticoagulant which is
isolated from the venom of the snake Agkistrodon rhodostoma (Merck
Index 1989, No. 664). A multiplicity of methods for its preparation
from snake venom have already been described (GB Patent 1,094,301,
GB Patent 1,177,506, GB Patent 1,293,793, U.S. Pat. No. 3,743,722,
U.S. Pat. No. 3,879,369, German Offenlegungsschrift 2,428,955,
German Offenlegungsschrift 2,734,427). These processes are
essentially based on chromatographic steps and yield the ancrod in
varying yield and purity.
The preparation of highly pure ancrod from snake venom has been
unsuccessful until now. A mixture of enzymes with ancrod as the
main component was always isolated which, depending on the
preparation, was contaminated with more or less foreign
proteins.
A way has now been found to prepare thrombin-like proteases from
snake venoms in highly pure form.
The invention relates to a process for purifying thrombin-like
proteases from snake venoms, which consists in
a. subjecting a protease crude product to a prepurification by
affinity chromatography or chromatography on a basic ion
exchanger,
b. subjecting the fraction containing the thrombin-like enzymes
thus obtained to chromatography on a weak cation exchanger or
separating it in the basic range by adsorption on glass and
c. subjecting the main component from step b to gel chromatography
or purifying this component in the acidic range by chromatography
on glass,
where, however, at least one of steps b and c comprises a
separation by adsorption or chromatography on glass.
The invention furthermore relates to thrombin-like proteases from
snake venoms in a purity of from 95 to 100%.
It is recommended for stage b to carry out the purification by
adsorption on glass and stage c with the aid of gel
chromatography.
In a particularly preferred embodiment of the invention,
purification both in stage b and in stage c is carried out by
adsorption or chromatography on glass.
Agmatine-, arginine- or heparin-Sepharose is particularly suitable
for the prepurification by affinity chromatography.
Basic ion exchangers suitable for the prepurification are
particularly DEAE-cellulose and DEAE-Sepharose.
Buffers which may be mentioned for ion exchange chromatography are,
in particular, tris-phosphate and sodium phosphate buffers.
Ion exchange chromatography is carried out at a pH of 5-9,
preferably of 6-8.5.
During the prepurification, approximately 70-80% of foreign
proteins and other constituents are removed from the crude
enzyme.
If the second step of the purification is carried out using a
cation exchanger, those suitable are the following weakly acidic
exchangers: CM-SEPHAROSE, pH 5-9, and AMBERLITE CG50, pH 5-9.
Chromatography on glass means that ancrod and related thrombin-like
enzymes as well as strongly basic proteases are bound to the glass
matrix at pHs of 7.5-9.0, preferably 8.0-8.5. About 60% of
especially acidic foreign proteins are washed from the column in
unbound form with the equilibration buffer (preferably
tris-phosphate or sodium phosphate buffer). Ancrod is eluted from
the glass fractionally in over 90% purity by increasing the ionic
strength of the buffer to 0.3-1.0 M by addition of sodium
chloride.
In the second purification step, the enzyme is concentrated to
approximately 90%.
For gel chromatography as a purification step c, suitable gels, in
particular, are: SEPHACRYL S-100HR, SUPERDEX, SEPHADEX, ULTROGEL
and SUPEROSE.
If chromatography on glass is selected for this purification step
c, in the acidic pH range from 4-6 basic foreign proteins are
adsorbed on the glass surface from the ancrod solution, while
ancrod can be eluted from the column directly in the equilibration
buffer in far over 95% purity. The desired ionic strength of the
buffer can be regulated by addition of a salt such as sodium
chloride.
The novel process is very particularly suitable for the preparation
of ancrod in pure form, which according to this purification
process is obtained in a purity of clearly over 95%.
EXAMPLE 1
a. Prepurification
3 g of dried venom of the Malayan pit viper were dissolved in 50 ml
of tris(hydroxymethyl)aminomethane(TRIS)-phosphate buffer pH 8.5,
insoluble cell constituents of the venom were centrifuged off and
the clear, yellow solution was applied to a chromatography column
which had a diameter of 1.6 cm and was packed up to a height of 30
cm with DEAE-SEPHAROSE-FF (Pharmacia). The thrombin-like enzymes of
the venom and the proteins having acidic character were bound to
the matrix. Chromatography was carried out at a flow rate of
150-200 ml/h. By washing the column at room temperature with about
300 ml of equilibration buffer (10 mM TRIS-phosphate buffer pH 8.5)
until the A.sub.280 nm value of the eluate had fallen below 0.5 and
further washing with 400 ml of 35 mM TRIS-phosphate buffer pH 6.0
until the A.sub.280 nm value of the eluate was <0.4, about
70-80% of the foreign proteins (based on the optical density of the
starting solution at 280 nm) were eluted. The main fraction
containing ancrod was eluted in 85% yield in 150-200 ml of 150 mM
TRIS-phosphate buffer pH 6.0.
b. Main purification
The eluate which contained the ancrod was concentrated to 20 ml by
means of ultrafiltration on a membrane having a nominal separation
limit of 10000 Daltons and rebuffered using a 100 mM TRIS-phosphate
buffer pH 8.0. This solution was applied to a column which had a
diameter of 1.6 cm and was packed up to a height of 15 cm with
BIORAN-CPG glass beads (Schott, pore diameter: 25-35 nm, particle
size: 30-60 .mu.m). By washing the column at room temperature with
300 ml of the 100 mM TRIS-phosphate buffer pH 8.0 and at a flow
rate of 250 ml per hour, about 60% of foreign proteins (based on
the optical density of the material applied at 280 nm) were eluted
from the column.
For elution, a 0.5 M sodium chloride solution which was buffered to
a pH of 8.0 using 100 mM TRIS-phosphate was employed. The eluate
was automatically collected in about 10 ml fractions. The
thrombin-like enzymes were eluted in one peak with a subsequent
tailing range. In order to obtain the main component (corresponds
to ancrod) in a form which contained at most 5% of thrombin-like
secondary components, in the transition from the main peak to the
tailing range individual fractions were investigated by means of
reverse-phase HPLC both for their specific fibrinogenase activity
and for their composition. Only fractions which had a specific
activity of greater than 1700 U/OD.sub.280 nm and which contained
less than 10% of secondary components in HPLC were combined with
the main peak (about 80 ml). The main component was obtained from
the BIORAN column in a purity of 96% and a yield of 72%.
c. Fine purification
The eluate containing the main component ancrod was concentrated to
2 ml by ultrafiltration on a YM 10 membrane (Amicon) and the
concentrate obtained was applied to a column which had a diameter
of 1.6 cm and was packed up to a height of 85 cm with SEPHACRYL
S-100 HR. The column had been equilibrated beforehand using a
buffer of 100 mM sodium chloride and 100 mM sodium phosphate which
had a pH of 6.9. Residual proteases and TRIS were separated from
ancrod by means of this gel chromatography. The yield in this step
was about 90%.
EXAMPLE 2
a. Prepurification
2.1 g of dried venom of the Malayan pit viper were dissolved in 50
ml of 35 mM TRIS-phosphate buffer pH 8.5, insoluble cell
constituents of the venom were centrifuged off and the clear,
yellow solution was applied to a chromatography column which had a
diameter of 1.6 cm, was packed up to a height of 30 cm with
DEAE-SEPHAROSE-FF (Pharmacia) and was equilibrated with the buffer
mentioned above. By washing the column at room temperature with 600
ml of equilibration buffer until the A.sub.280 nm value of the
eluate was <0.2, approximately 70% of the foreign proteins
(based on the optical density of the starting solution at 280 nm)
were eluted. The main fraction was eluted in 90-100% yield using
200-250 ml of 150 mM TRIS-phosphate buffer pH 6.0.
b. Main purification
The eluate was concentrated to 20 ml as in Example 1 and rebuffered
using a 50 mM Na phosphate buffer pH 8.5. This solution was applied
to the BIORAN-CPG glass column (diameter: 1.6 cm, height: 15 cm,
pore diameter: 25-35 nm, particle size: 30-60 .mu.m). By washing
the column at room temperature with 300 ml of the 50 mM Na
phosphate buffer pH 8.5 and at a flow rate of 250 ml per hour,
about 60% of foreign proteins (based on the optical density of the
material applied at 280 nm) were eluted from the column.
For elution of the thrombin-like enzymes, a 1 M sodium chloride
solution which had been buffered to a pH of 8.0 using 50 mM
Na-phosphate was employed. Using 150 ml of buffer, about 80% of the
units of enzyme applied were eluted.
c. Fine purification
The eluate was concentrated to 20 ml as above and rebuffered using
a 50 mM Na phosphate buffer which had a pH of 5.0. This solution
was applied to a column which also had a diameter of 1.6 cm and was
packed up to a height of 15 cm with BIORAN-CPG glass which,
however, had a pore diameter of 90-110 nm and a particle size of
30-60 .mu.m. At pH 5.0, only basic proteins and the secondary
components were bound to the BIORAN glass, while the main component
ancrod was eluted from the column using the equilibration buffer in
a purity of approximately 100% and in about 80-90% yield (based on
the units applied).
EXAMPLE 3
a, b. Pre- and main purification
2.1 g of dried venom of the Malayan pit viper were prefractionated
on DEAE-SEPHAROSE analogously to Example 2, and the eluate was
concentrated to 20 ml analogously to Example 1 and rebuffered using
a 40 mM TRIS-phosphate buffer pH 6.2. This solution was applied to
a chromatography column which had a diameter of 1.6 cm, [lacuna]
packed up to a height of 20 cm with CM-SEPHAROSE-FF (Pharmacia) and
was equilibrated with the abovementioned buffer. By washing the
column at room temperature with 300 ml of the equilibration buffer
and at a flow rate of 250 ml/h, about 60% of foreign proteins
(based on the optical density of the material applied at 280 nm)
were eluted from the column.
For elution of the thrombin-like enzymes, analogously to Example 2
a 1 M sodium chloride solution which was buffered to a pH of 8.0
using 50 mM Na phosphate was employed. Using 150 ml of buffer,
about 80% of the units of thrombin-like enzymes applied were
eluted.
c. Fine purification
The fine purification of ancrod was carried out analogously to
Example 2 on BIORAN-CPG glass (pore diameter about 100 nm; particle
size 30-60 .mu.m) using a 50 mM phosphate buffer pH 5.0. Ancrod was
eluted from the column in over 95% purity and in about 85% yield
(based on the units applied).
* * * * *